WO2015067161A1

WO2015067161A1 - Phospholipase c mutant and use thereof

Info

Publication number: WO2015067161A1
Application number: PCT/CN2014/090213
Authority: WO
Inventors: 许骏; 宣姚吉; 李金敏
Original assignee: 丰益(上海)生物技术研发中心有限公司
Priority date: 2013-11-07
Filing date: 2014-11-04
Publication date: 2015-05-14
Also published as: CN104630174B; CN104630174A; US10144919B2; US20160362665A1

Abstract

The present application provides a wild type phosphatidylcholine specificity phospholipase C (PLC) mutant of Bacillus cereus, the related mutations comprising asparagine at position 63 mutating to another amino acid; also comprising mutation of arginine to histidine at position 20 and of alanine to aspartic acid at position 83. The present application also provides a nucleic acid molecule encoding said mutant, a vector containing said nucleic acid molecule, and a cell containing said nucleic acid molecule or vector. The present application also provides uses of said mutant, nucleic acid molecule vector, and cell.

Description

Phospholipase C mutant and use thereof

Field of invention

The present application relates to phosphatidylcholine-specific phospholipase C mutants and uses thereof.

Background of the invention

Degumming is an important step in oil refining. The traditional hydration degumming method has high economic cost, high material energy consumption and heavy environmental pollution. Therefore, in recent years, many work has been devoted to degumming enzymatic degumming for degumming in oil refining. Compared with the traditional method, enzymatic degumming can improve economic efficiency, achieve energy saving and emission reduction, less pollution to the ecological environment, and has greater advantages in environmental protection, economy and quality. One enzyme used in the degumming of fats is a phospholipase. Phospholipase C (PLC) exhibits greater advantages than other degumming enzymes, for example, increasing the yield of glycidyl ester (DAG) and reducing the loss of oil.

Phosphatidylcholine-specific phospholipase C (BC-PC-PLC) of Bacillus cereus is one of the earliest studied phospholipase Cs. BC-PC-PLC is 283 amino acids in length and contains a 24 amino acid signal peptide and a 14 amino acid leader peptide. The mature peptide is 245 amino acids (see, for example, Johansen, T., Holm, T., Guddal, PH, Sletten, K., Haugli, FB, Little, C. (1988). "Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus." Gene 65(2): 293-304).

The crystal structure of BC-PC-PLC has been reported to consist of multiple helical domains with a catalytic site of 55 aspartic acid and at least three Zn ²⁺ binding sites (see, for example, Hough., E., Hansen, LK, Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., Derewenda, Z. (1989) "High-resolution (1.5) A) crystal structure of phospholipase C from Bacillus cereus. "Nature. 338:357-60). There are few studies on heterologous expression of BC-PC-PLC, and it has been reported to express BC-PC-PLC in Bacillus subtilis and Pichia pastoris (see, for example, Durban, MA, Silbersack, J., Schweder, T., Schauer, F., Bornscheuer, UT (2007) High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 74(3): 634-639; and Seo, KH, Rhee JI (2004) High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization. Biotechnol Lett 26(19): 1475-1479).

There is a need in the art for improved (e.g., higher enzymatic activity) phospholipase C.

Summary of invention

In a first aspect, the application provides a polypeptide having phosphatidylcholine-specific phospholipase C activity, comprising a mutated amino acid sequence set forth in SEQ ID No: 2, or an active fragment thereof, wherein the mutation comprises SEQ The asparagine at position 63 of the amino acid sequence shown by ID No: 2 was mutated.

In some embodiments, the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S), alanine (A), phenylalanine (F), histidine (H) ), lysine (K), arginine (R), tryptophan (W), tyrosine (Y), cysteine (C), aspartic acid (D), glutamic acid ( E), glycine (G), isoleucine (I), leucine (L), methionine (M), glutamine (Q), threonine (T) or valine (V). In some embodiments, the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S).

In some embodiments, the mutation further comprises the substitution of the arginine at position 20 of the amino acid sequence of SEQ ID No: 2 with histidine and the alanine at position 83 with aspartic acid.

In some embodiments, the amino acid sequence of the polypeptide comprises a SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44 , amino acid sequence of 46 or 48 or selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48 The amino acid sequence consists of.

In some embodiments, the amino acid sequence of the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID No: 12.

In a second aspect, the application provides a nucleic acid molecule encoding the polypeptide of the first aspect.

The application also provides a nucleic acid molecule comprising SEQ ID No: 7, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45 or A nucleic acid sequence of 47.

The application also provides a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID No:11.

In a third aspect, the application provides a vector comprising the nucleic acid molecule of the second aspect.

In some embodiments, the vector is an expression vector. In some embodiments, the vector is designed for expression in a eukaryotic or prokaryotic cell. In some embodiments, the vector is designed for expression in a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.

In a fourth aspect, the application provides a cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell.

In a fifth aspect, the present application provides the phospholipase C produced by the cell of the fourth aspect.

In a sixth aspect, the present invention provides the polypeptide of the first aspect, or the polypeptide encoded by the nucleic acid molecule of the second aspect, or the vector encoded by the vector of the third aspect, or the cell expression of the fourth aspect The polypeptide or the phospholipase C of the fifth aspect is used as the phosphatidylcholine-specific phospholipase C.

In some embodiments, the use is in a grease degumming process.

In a seventh aspect, the present application provides the polypeptide of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the cell of the fourth aspect, for use in preparing a degumming enzyme .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a MM- of wild-type BC-PC-PLC Pichia pastoris expressing strains G15 and 1-3 obtained in Example 1. The results of the yolk selection plate showed that the top 4 clones were G15 and the lower 6 clones were 1-3, wherein the strain was cultured at 30 ° C for three days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Fig. 2 is a graph showing the results of the MM-yolk screening panel of the mutant strain 7-3-3 obtained in Example 2, in which the strain was cultured at 30 ° C for 2 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Fig. 3 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-R20H obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Fig. 4 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-N63S obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Fig. 5 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-A83D obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Fig. 6 is a graph showing the results of the MM-yolk screening panel of the BC-PC-PLC point mutation Pichia pastoris expression strain PLC-R20HN63SA83D obtained in Example 4, wherein the strain was cultured at 30 ° C for 3 days. The name of the corresponding strain is indicated directly below the white sedimentation circle.

Figure 7 shows the ratio of the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1 and the four BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 4. Enzyme activity.

Figure 8 shows the shaking of the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1 and the four BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 4. SDS-PAGE electropherogram of the bottle fermentation broth.

Figure 9 shows the shake flask fermentation broth protein concentration of the 17 BC-PC-PLC point mutation Pichia pastoris expression strains obtained in Example 6.

Fig. 10 is a view showing the SDS-PAGE electrophoresis of the shake flask fermentation broth of the partial BC-PC-PLC point mutation Pichia pastoris expression strain obtained in Example 6.

Figure 11 shows the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1, the BC-PC-PLC point mutation Pichia pastoris expression strain 6-6 obtained in Example 6 and 7-6 and the specific enzyme activities of the 18 BC-PC-PLC point mutant Pichia pastoris expressing strains obtained in Example 8 under the reaction conditions of 37 ° C and 60 ° C.

Figures 12 and 13 show the wild type BC-PC-PLC Pichia pastoris expression strains G15 and 1-3 obtained in Example 1, respectively, and the BC-PC-PLC point mutation Pichia pastoris expression strain 6 obtained in Example 6 The thermal stability of the BC-PC-PLC point mutant Pichia pastoris expressing strains obtained in -6 and 7-6 and in Example 8.

A detailed description

Definition

The phosphatidylcholine phospholipase C described herein is synonymous with phosphatidylcholine-preferring phospholipase C and is also readily available to those skilled in the art. Understand. A shorthand PC-PLC is used herein to mean phosphatidylcholine-specific phospholipase C or phosphatidylcholine-preferred phospholipase C. An example of a phosphatidylcholine-specific phospholipase C for use herein is the phosphatidylcholine-specific phospholipase C of Bacillus cereus, denoted herein by the abbreviated BC-PC-PLC. It should be understood that, herein, BC-PC-PLC may represent the wild-type phosphatidylcholine-specific phospholipase C of Bacillus cereus, which may also be referred to in the present application based on the wild-type phosphatidylcholine-specific phospholipase C. The mutant obtained.

Where reference is made herein to the numerical representation of the amino acid position, the number is referenced to the amino acid position in SEQ ID No: 2, and SEQ ID No: 2 is the wild type phosphatidylcholine-specific phospholipase C of Bacillus cereus The amino acid sequence of the mature peptide.

One-letter or three-letter abbreviations for internationally accepted amino acids are used herein.

The terms "polypeptide", "peptide" and "protein" as used herein are used interchangeably and refer to a polymer formed by the joining of a plurality of amino acids by peptide bonds. Amino acids can be naturally occurring or synthetic analogs.

The terms "nucleic acid" and "polynucleotide" as used herein are used interchangeably and include, but are not limited to, DNA, RNA, and the like. Nucleotides can be naturally occurring or synthetic analogs.

The cells herein may be eukaryotic cells or prokaryotic cells such as, but not limited to, bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells.

Detailed description of the technical solution

The present application provides phospholipase C mutants obtained by mutation screening methods in molecular biology and uses thereof. Specifically, the phospholipase C may be phosphatidylcholine-specific phospholipase C (PC-PLC). More specifically, the phosphatidylcholine-specific phospholipase C may be phosphatidylcholine-specific phospholipase C (BC-PC-PLC) of Bacillus cereus.

In some embodiments, the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to alanine (A), cysteine (C), aspartic acid (D), glutamine Acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M) ), glutamine (Q), arginine (R), threonine (T), valine (V), tryptophan (W) or tyrosine (Y). In some embodiments, the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S).

In some embodiments, the amino acid sequence of the polypeptide comprises a SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44 , amino acid sequence of 46 or 48 or selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48 The amino acid sequence consists of. In some embodiments, the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44 The amino acid sequence of 46 or 48, that is, the amino acid sequence shown by SEQ ID No: 2 differs only in the amino acid mutation at position 63 described above.

In some embodiments, the amino acid sequence of the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID No: 12. In some embodiments, the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID No: 12, ie, differs from the amino acid sequence set forth in SEQ ID No: 2 only in the 20th position described above. The mutation was histidine (H), the mutation at position 63 was serine (S), and the mutation at position 83 was aspartic acid (D).

In some embodiments, the amino acid sequence of the polypeptide is the same as the length of the amino acid sequence set forth in SEQ ID No: 2.

In some embodiments, the amino acid sequence of the polypeptide has a length greater than the amino acid sequence set forth in SEQ ID No: 2. In some embodiments, the polypeptide further comprises a signal peptide and/or a leader peptide. As described in the "Background of the Invention" section, the wild type phosphatidylcholine-specific phospholipase C of Bacillus cereus comprises a 24 amino acid signal peptide and a 14 amino acid leader peptide, and thus the polypeptide of the present application may also comprise the same Or other signal peptides and/or leader peptides. In some embodiments, the signal peptide is an alpha factor signal peptide. Those skilled in the art will appreciate that the polypeptides of the present application may also include other functional elements such as, but not limited to, tag elements for isolation and purification (eg, histidine tags), selection elements (eg, based on antibiotic selection or fluorescent selection) (eg green fluorescent protein, GFP)) and the like.

In some embodiments, the polypeptide has an amino acid sequence that is less than the amino acid sequence set forth in SEQ ID No: 2. In related embodiments, the polypeptide may comprise a mutated active fragment of the amino acid sequence set forth in SEQ ID No: 2, such as SEQ ID Nos: 8, 12, 14, 16, 18, 20, 22, 24, 26, An active fragment of the amino acid sequence shown at 28, 30, 32, 34, 38, 40, 42, 44, 46 or 48. The term "active fragment" as used herein denotes a part of a wild-type phosphatidylcholine-specific phospholipase C or a phosphatidylcholine-specific phospholipase C mutant of the present application which still retains phosphatidylcholine-specific phospholipids. Activity of enzyme C. The structural and functional sites of the wild-type phosphatidylcholine-specific phospholipase C of Bacillus cereus are known in the art, and thus those skilled in the art can readily prepare active fragments of the polypeptide retaining the above mutation, For example, keep a fragment of a functional domain. It has been reported in the literature that the active sites of the PLC are Glu4, Asp55, Tyr56, Glu146, Ser64, Thr65, Phe66, Phe70, Ile80, Thr133, Asn134, Leu135, Ser143 (see, for example, Hough., E., Hansen, LK, Birknes , B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., Derewenda, Z. (1989) "High-resolution (1.5A) crystal structure of phospholipase C from Bacillus cereus. "Nature. 338:357-60", according to the crystal structure of the PLC, the 8th and 9th alpha helices are amino acids 140-153 and 154-157 The amino acid, the 10th alpha helix is amino acids 171-186, so the predicted active fragment is 1-170 amino acids.

The present application also contemplates functional variants of the polypeptides described in the first aspect. In some embodiments, the functional variant is a conservative substitution variant. "Conservative substitution" refers to a change in the amino acid composition of a protein that does not significantly alter the activity of the protein. Thus "conservative substitution" of a particular amino acid sequence refers to the substitution of those amino acids that are not critical to protein activity, or with similar properties (eg, acidic, basic, positively or negatively charged, polar or non-polar, etc.) The other amino acids replace the amino acids so that even the substitution of the key amino acids does not significantly alter the activity. Conservative substitution representatives that provide functionally similar amino acids are well known in the art. For example, each of the 6 groups in the table below includes amino acids that are conservatively substituted with each other:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) Phenylalanine (F), tyrosine (Y), and tryptophan (W).

See also Creighton, Proteins: Structures and Molecular Properties, W. H. Freeman and Company, New York (2nd Ed., 1992).

In some embodiments, the functional variant and the parent sequence (eg, SEQ ID No: 8, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40 The amino acid sequence of 42, 42, 44 or 48) has an identity or similarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher.

In a second aspect, the application provides a nucleic acid molecule encoding the polypeptide of the first aspect. The present application contemplates different nucleic acid molecules that are obtainable due to the degeneracy of the genetic code or the preference of different species for codons.

The application also provides a nucleic acid molecule comprising SEQ ID No: 7, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45 or 47 nucleic acid sequence.

It will be understood that the nucleic acid molecules of the present application may comprise not only the coding sequences of BC-PC-PLC mature peptide mutants, but also other nucleic acid sequences. In some embodiments, the additional nucleic acid sequence is a nucleic acid sequence encoding a signal peptide and/or a leader peptide. In some embodiments, the additional nucleic acid sequence is a nucleic acid sequence encoding a tagging element (eg, a histidine tag) for isolation and purification, encoding a selection element (eg, based on antibiotic selection or fluorescent selection (eg, green fluorescent protein, GFP) )) The nucleic acid sequence. Those skilled in the art will appreciate that the other nucleic acid sequences may also be regulatory sequences required for transcription and/or translation, such as promoters, enhancers, and the like.

In a third aspect, the application provides a vector comprising the nucleic acid molecule of the second aspect. In some embodiments, the vector is an expression vector. In some embodiments, the vector is designed for use in a eukaryotic or prokaryotic cell expression. In some embodiments, the vector is designed for expression in a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell. In some embodiments, the vector is a plasmid. Suitable eukaryotic or prokaryotic vectors are well known to those skilled in the art, and a variety of parent carriers are commercially available. Examples of vectors include, but are not limited to, a variety of vectors used in embodiments of the present application.

In a fourth aspect, the application provides a cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell, or a plant cell. In some embodiments, the cell is a pichia pastoris cell. In some embodiments, the cell is a Bacillus subtilis cell. With respect to a cell comprising a nucleic acid molecule of the present application, the nucleic acid molecule can be located extrachromosomally (e.g., in a vector) or can be integrated into the chromosome of a host cell. Techniques for integrating a nucleic acid molecule into the chromosome of a host cell and introducing the vector into the host cell by transformation or transfection are well known to those skilled in the art.

In a fifth aspect, the present application provides the phospholipase C produced by the cell of the fourth aspect. Techniques for producing a polypeptide or protein of interest using genetically engineered host cells are well known to those skilled in the art.

In a sixth aspect, the present invention provides the polypeptide of the first aspect, or the polypeptide encoded by the nucleic acid molecule of the second aspect, or the vector encoded by the vector of the third aspect, or the cell expression of the fourth aspect The polypeptide or the phospholipase C of the fifth aspect is used as the phosphatidylcholine-specific phospholipase C. In some embodiments, the use is in a grease degumming process. The use of phosphatidylcholine-specific phospholipase C in a grease degumming process is known in the art. Phospholipase C is capable of hydrolyzing the colloidal phospholipids in the oil to form a hydrophilic phosphate moiety and a lipophilic DAG. The hydrophilic portion is carried away by water to remove the colloidal fraction, and DAG increases the oil yield. For example, in the enzymatic degumming process, the feedstock oil is heated to 60 ° C, the phospholipase C solution is added, and after high-speed shear mixing, the reaction is stirred in the reactor for 2 h, and then the aqueous phase and the oil phase are separated by centrifugation.

In a seventh aspect, the present application provides the polypeptide of the first aspect, or the nucleic acid molecule of the second aspect, or the vector of the third aspect, or the cell of the fourth aspect, for use in preparing a degumming enzyme . Methods for preparing degumming enzymes using polypeptides, nucleic acid molecules, vectors or transformed cells are well known in the art. Taking the fermentation method as an example, the DNA sequence of the isolated phospholipase C sequence can be transformed into a host cell (for example, Pichia pastoris) by an expression vector, and subjected to large-scale fermentation culture in a fermenter, and the fermentation liquid is obtained by filtration, and then passed through ultrafiltration. The corresponding buffer is replaced to remove the higher concentration of salt ions in the fermentation broth, and a common stabilizer (such as glycerin) and metal ion zinc (added as zinc sulfate) are added to the ultrafiltrate.

It is to be understood that the foregoing detailed description is only to be understood as Those skilled in the art will be able to make various modifications and variations to the described embodiments.

Example

The following examples are provided to further describe the present application without any limitation.

Experimental Materials

The main materials used in the examples of the present application are as follows:

1. Strain and plasmid

Strains: Pichia pastoris GS115 (Invitrogen, Cat. No. C181-00), Escherichia coli DH5a (TAKARA, Cat. No. D9057A).

Plasmid: pPIC-9k (Invitrogen, Cat. No. V17520), pAO815 plasmid (Invitrogen, Cat. No. V18020), pAO-PLC plasmid and pAOmu-PLC plasmid were constructed by the inventors of the present application, as described in detail below.

2. Medium and solution

LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.

LB solid medium: 1.5% agar was added to the LB liquid medium.

YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

YPD solid medium: 2% agar was added to the LB liquid medium.

MGYS solid medium: 1.34% yeast nitrogen source base (YNB) (containing ammonium sulfate, no amino acid), 1% glycerol, 1 M sorbitol, 4 x 10 ^-5 % D-biotin, 2% agar.

MM-yolk screening medium: 1.34% yeast nitrogen source base (YNB) (containing ammonium sulfate, no amino acid), 4 × 10 ^-5 % D-biotin, 0.5% methanol (added after sterilization), 2% egg yolk Liquid, 2% agar.

Preparation of egg yolk solution: Take one fresh egg, wipe it with 75% alcohol, knock the eggshell with sterile scorpion to make the egg white flow out, rinse the egg yolk with sterile water twice, and add it to a triangular bottle containing 80ml of sterile water. Mix well to obtain 20% egg yolk.

BMGY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) (ammonium sulfate, no amino acid), 1% glycerol, 4×10 ^-5 % D-biotin, 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 6.0).

BMMY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source base (YNB) (ammonium sulfate, no amino acid), 0.5% methanol (added after sterilization), 4×10 ^-5 % D-biotin (added after sterilization), 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 6.0).

3. Reagents for detecting PLC activity by molybdenum blue method:

PLC reaction solution: 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl ₂

CIAP reaction solution: 50 mM Tris-HCl pH 9.0, 10 mM MgCl ₂ , 1 U CIAP (purchased from Bao Bioengineering (Dalian) Co., Ltd.)

Molybdenum blue color reaction solution: 100 μl CIAP reactant, 0.2% ascorbic acid, 0.1% ammonium molybdate (prepared with 30% H ₂ SO ₄ )

4. Protein concentration detection reagent:

Modified Bradford Method Protein Concentration Kit (purchased from Shanghai Shenggong Bioengineering Co., Ltd.)

5. Enzyme:

Restriction enzymes HindIII, EcoRI, AatII (purchased from New England Biotechnology (Beijing) Co., Ltd.)

PCR enzyme: TaKaRa Taq,

DNA polymerase (purchased from Bao Bioengineering (Dalian) Co., Ltd.)

T4DNA ligase (purchased from Enzyme Co., Ltd.)

Example 1: Construction of wild-type BC-PC-PLC Pichia pastoris expression strain

The DNA sequence of BC-PC-PLC was designed according to the mature peptide sequence of phosphatidylcholine-specific phospholipase C of Bacillus cereus (PDB ID: 1AH7) and Pichia codon preference (SEQ ID No: 1). And fused the alpha factor signal peptide sequence at its front end (the DNA sequence of which is derived from the commercial Pichia pastoris expression vector pPIC-9k, positions 8-274 of SEQ ID No: 3) and the Kozak sequence of Pichia pastoris (SEQ. ID No: 3, 1st to 7th), the α-BC-PC-PLC DNA sequence (SEQ ID No: 3) was finally obtained.

The α-BC-PC-PLC DNA sequence was supplied to Shanghai Shenggong Biotechnology Co., Ltd. for whole gene synthesis, and the cloning vector pGEM-T-PLC containing the α-BC-PC-PLC DNA sequence was obtained. Use this carrier as a template, use

The DNA polymerase and the primer pair AmPLC-3/AmPLC-4 were amplified by PCR to obtain a PLC fragment.

Using the pPIC-9k expression vector as a template

The DNA polymerase and the primer pair AmPLC-1/AmPLC-2 were amplified by PCR to obtain an AOX1 promoter fragment (PAOX1).

Using overlapping primers, AmPLC-1/AmPLC-4 and

DNA polymerase to obtain a PAOX1+PLC fusion fragment.

The PAOX1+PLC fusion fragment was cloned into the pAO815 vector using AatII and EcoRI cleavage sites to obtain the expression vector pAO-PLC. The pAO-PLC was linearized with SalI and the gel was recovered to an approximately 8.5 kb fragment. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and the linearized pAO-PLC fragment was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked and suspended in 5 μl of sterile water. 0.5 μl was inoculated on the MM-yolk screening plate. After culturing at 30 ° C for 3 days, a positive clone of a white sediment circle was observed around the cells. The expressed phosphatase C can decompose lecithin into phosphatidylcholine and water-insoluble diglyceride, and the white sinking ring around the colony of MM-yolk screening plate belongs to the above reaction. When the colony has a large amount of phospholipase C secretion or the secreted phospholipase C is higher than the enzyme activity, the white precipitate circle formed around the colony is relatively large.

Two positive strains were screened, as shown in Fig. 1, which were designated as G15 and 1-3, respectively. The white precipitate circle of G15 was smaller, and the white precipitate circle of 1-3 was larger.

Example 2: Construction and screening of BC-PC-PLC mutant library

Using pAO-PLC vector as template

DNA polymerase and primer pair AmPLC-1/AOXH-2 were amplified by PCR to obtain a fragment of about 900 bp. Using pAO-PLC vector as template

DNA polymerase and primer pair AOXH-3/AmPLC-4, amplified by PCR to obtain a fragment of about 1.1 kb. The approximately 900 bp fragment obtained by the previous two-step PCR and the about 1.1 kb fragment were mixed as a template for the third step PCR, using primer pair AmPLC. -1/AmPLC-4 and

DNA polymerase was amplified by PCR to obtain a fragment of about 1.9 kb.

The approximately 1.9 kb fragment was cloned into pAO-PLC by AatII and EcoRI cleavage sites to obtain pmAO-PLC. In pmAO-PLC, a HindIII restriction site in pAO-PLC was mutated to retain only one HindIII restriction site located at the 5' end of the BC-PC-PLC sequence, enabling the use of HindIII and EcoRI for BC The mutated fragment of the -PC-PLC was cloned into pmAO-PLC.

The pAO-PLC vector was used as a template, and the error-prone PCR was performed on EPPLC-1/EPPLC-2 using TaKaRa Taq enzyme and primers (addition of 0.3 mM MnCl ₂ at the time of PCR) to obtain a mutant amplicon fragment of about 755 bp in size. set. The obtained fragment was cloned into pmAO-PLC by HindIII and EcoRI digestion sites, and the resulting vector was transformed into Escherichia coli DH5α strain, and a total of 1 × 10 ⁴ BC-PC-PLC mutants were obtained.

Each 1 × 10 ³ BC-PC-PLC mutants were washed with 2 ml of sterile water into 8 ml of LB liquid medium (containing 100 μg/ml ampicillin), and cultured at 37 ° C for 4 hours. The plasmid was extracted, linearized with SalI, and a fragment of about 8.5 kb was recovered. 500 ng of vector (using as little DNA as possible to ensure that most positive transformants contain a single copy of the PLC gene) was transformed into competent cells of the Pichia pastoris GS115 strain by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days to obtain a Pichia pastoris mutant library of BC-PC-PLC. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the white precipitate circle in the mutant and G15 was compared with G15 as a control. A final mutant strain with a white precipitated ring larger than G15 was designated for screening, designated 7-3-3, as shown in Figure 2.

Example 3: BC-PC-PLC mutant sequence analysis

The 7-3-3 strain was inoculated into 3 ml of YPD liquid medium, cultured at 30 ° C overnight, and genomic DNA was extracted. Using the genomic DNA of 7-3-3 strain as a template, use

The DNA polymerase and the primers were subjected to PCR amplification of AOX1-5/AOX1-3 to obtain the DNA sequence of BC-PC-PLC in the 7-3-3 strain. The obtained sequence was sent to Shanghai Shenggong Bioengineering Co., Ltd., and the primers were used to sequence AOX1-5/AOX1-3. The DNA sequencing results of 7-3-3 BC-PC-PLC are shown in SEQ ID No: 4. After comparison, it was found that compared with SEQ ID No: 3, 7 bases in SEQ ID No: 4 were mutated, including three sense mutations, respectively, the 59th G mutation was A, making The arginine at position 20 is mutated to histidine (CGT→CAT); the mutation at position 188 is G, such that the 63th asparagine is mutated to serine (AAC→AGC); the 248th C is mutated to A, making The alanine at position 83 was mutated to aspartic acid (GCC→GAC).

Example 4: Construction and screening of BC-PC-PLC single-point mutant Pichia pastoris expression strain

Example 4.1: Construction and Screening of PLC-R20H

Using pAO-PLC vector as template

DNA polymerase and primer pair EPPLC-1/20RH-2 were amplified by PCR to obtain a fragment of about 78 bp. Using pAO-PLC vector as template

DNA polymerase and primer pair 20RH-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 707 bp. The approximately 78 bp fragment obtained by the previous two-step PCR and the approximately 707 bp fragment were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and

DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.

The about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain a pmAO-PLC-R20H vector. The pmAO-PLC-R20H was linearized with SalI, and the 8.5 kb fragment was recovered by gel. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-R20H was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the mutant and the G15 and 1-3 white sedimentary circles were compared with G15 and 1-3 as shown in Fig. 3. A negative clone was found on the selection plate without a white sedimentary circle, ie, the PLC gene fragment was not successfully transferred into the genome of the clone. In selecting mutant positive clones for subsequent experiments, it was preferred to identify those clones with the largest proportion of all positive clones and the same or similar white precipitate circle size, and then randomly select one PLC-R20H designated for subsequent experiments. The mutant clones thus selected often contain a single copy of the PLC gene, which facilitates reducing the effect of differences in expression levels on enzyme activity comparison.

Example 4.2: Construction and screening of PLC-N63S

Using pAO-PLC vector as template

DNA polymerase and primer pair EPPLC-1/63NS-2, amplified by PCR to obtain a fragment of about 207 bp. Using pAO-PLC vector as template

DNA polymerase and primer pair 63NS-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 576 bp. The 207 bp fragment and the approximately 576 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and

DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.

The about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain pmAO-PLC-N63S vector, pmAO-PLC-N63S was linearized with SalI, and the 8.5 kb fragment was recovered by gel. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-N63S was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the mutant and the G15 and 1-3 white sedimentation circles were compared with G15 and 1-3 as shown in Fig. 4. The mutant positive clone PLC-N63S was used for subsequent experiments as described in Example 4.1.

Example 4.3: Construction and Screening of PLC-A83D

Using pAO-PLC vector as template

DNA polymerase and primer pair EPPLC-1/83AD-2 were amplified by PCR to obtain a fragment of about 266 bp. Using pAO-PLC vector as template

DNA polymerase and primer pair 83AD-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 520 bp. The 266 bp fragment and the approximately 502 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using primer pairs EPPLC-1/EPPLC-2 and

DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.

The about 755 bp fragment was cloned into pmAO-PLC by HindIII and EcoRI cleavage sites to obtain pmAO-PLC-A83S vector. The pmAO-PLC-A83S was linearized with SalI, and the 8.5 kb fragment was recovered by gel. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-A83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the mutant and the G15 and 1-3 white sedimentary circles were compared with G15 and 1-3 as shown in Fig. 5. The mutant positive clone PLC-A83D was used for subsequent experiments as described in Example 4.1.

Example 4.4: Construction and screening of PLC-R20HN63SA83D

Using the genomic DNA of 7-3-3 strain as a template, use

DNA polymerase and primer pair EPPLC-1/EPPLC-2, amplified by PCR to obtain a fragment of about 755 bp.

This fragment was cloned into pmAO-PLC by HindIII and EcoRI to obtain a pmAO-PLC-R20HN63SA83D vector. The pmAO-PLC-R20HN63SA83D was linearized with SalI, and the 8.5 kb fragment was recovered by gel. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 500 ng of linearized pmAO-PLC-R20HN63SA83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the mutant and the G15 and 1-3 white precipitation circles were compared with G15 and 1-3 as shown in Fig. 6. The mutant positive clone PLC-R20HN63SA83D was used for subsequent experiments as described in Example 4.1.

The white precipitate on the screening plate of the PLC-N63S mutant was substantially equivalent to the white precipitate of the PLC-R20HN63SA83D clone and was significantly larger than the white precipitates of G15 and 1-3. The size of the PLC-R20H and PLC-A83D mutants on the white precipitation circle is approximately the same as the white precipitation circle size of G15 and 1-3. Thus, it was confirmed that the mutation of amino acid 63 from asparagine to serine can enhance the enzyme activity of BC-PC-PLC.

Example 5: Shake flask fermentation of BC-PC-PLC mutant strain

G15, 1-3, PLC-R20H, PLC-N63S, PLC-A83D and PLC-R20HN63SA83D strains were first activated in liquid YPD, then inoculated in BMGY medium, and cultured overnight at 30 ° C with shaking at 220 rpm. The culture was transferred to BMMY medium, the initial OD ₆₀₀ of 6.

First, induction with 2% methanol, 1% methanol was added after 24h and 32h, and 1% methanol was added after 48h and 56h, and samples were taken at 72h. The obtained sample was subjected to ultrafiltration desalting using an ultrafiltration tube having a molecular weight cut off of 10 kDa. The treated sample was added to a buffer (20 mM citric acid-sodium citrate buffer (pH 6.6), 30% glycerol, 0.3% ZnSO ₄ ·7H ₂ O).

10 μl of the fermentation broth was added to 190 μl of PLC reaction solution (containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl ₂ ), and incubated at 37 ° C for 30 min with shaking. After the incubation, 100 μl of chloroform was added and shaken, and centrifuged at 12,000 rpm for 2 min. 80 μl of the supernatant was taken, and 20 μl of CIAP reaction solution (containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl ₂ , 1 U CIAP) was added, and the mixture was incubated at 37 ° C for 1 h with shaking.

After incubation, 25 μl of the reaction was taken, and 975 μl of molybdenum blue color developing solution (containing 0.2% ascorbic acid, 0.1% ammonium molybdate) was added, and the mixture was incubated at 37 ° C for 10 min with shaking. The absorbance of the sample was measured at a wavelength of 700 nm, and the PLC activity of each fermentation broth sample was calculated. The protein concentration of the shake flask fermentation broth of G15, 1-3, PLC-R20H, PLC-N63S, PLC-A83D and PLC-R20HN63SA83D strains was determined by Bradford reagent, and the results were as follows: G15, 1-3, PLC-R20H, PLC -N63S, PLC-A83D and PLC-R20HN63SA83D specific enzyme activity. As shown in Fig. 7, the specific enzyme activity of PLC-N63S and PLC-R20HN63SA83D is about 4 times that of wild type, and the specific enzyme activity of PLC-R20H and PLC-A83D is equivalent to wild type, which proves that the 63th asparagine mutation is Serine is a key mutation site that is increased in activity over enzyme activity.

The protein amount of the shake flask fermentation broth of G15, 1-3, PLC-R20H, PLC-N63S, PLC-A83D and PLC-R20HN63SA83D strains was adjusted to the same amount for SDS-PAGE electrophoresis, and the results are shown in Fig. 8. The PLC-N63S PLC protein band is slightly less intense than the G15 PLC protein band, so the specific enzyme activity of PLC-N63S is at least 4 times that of G15.

Example 6: Construction of high-yield strains of PLC-N63S and PLC-R20HN63SA83D and shake flask fermentation

The pmAO-PLC-N63S and pmAO-PLC-R20HN63SA83D were linearized with SalI, and the gel recovered approximately 8.5 kb fragment. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and 2 μg of linearized pmAO-PLC-R20HN63SA83D was transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. Eleven white precipitates were selected from the 100 PLC-N63S transformants and designated as 6-1 to 6-11. Similarly, the six largest transformants of the white precipitate were selected from 100 PLC-R20HN63SA83D transformants and designated 7-1 to 7-6.

The strains 6-1 to 6-11 and 7-1 to 7-6 were firstly activated in liquid YPD, then inoculated into BMGY medium, and cultured overnight at 30 ° C with shaking at 220 rpm. The culture was transferred to BMMY medium, the initial OD ₆₀₀ of 6.

The protein concentration of the shake flask fermentation broth of 6-1 to 6-11 and 7-1 to 7-6 strains was measured with Bradford reagent, and the results are shown in Fig. 9.

Among the 6-1 to 6-11 strains, the highest concentration of fermentation broth protein was 6-6 strain, the fermentation broth protein concentration was 0.108 mg/ml, and the highest concentration of fermentation broth protein in 7-1 to 7-6 was 7- 6 strain, the fermentation broth protein concentration was 0.238 mg / ml. SDS-PAGE electrophoresis was performed on several strains with higher protein concentrations in the two strains, and the amount of band protein in the PLC was compared. The results are shown in Fig. 10. The intensity of the PLC target bands of 7-5 and 7-6 is higher than 6 The amount of the PLC target band of -2, 6-3, 6-6, 6-9, 6-11. Thus, the R20H and A83D mutations in the PLC-R20HN63SA83D mutant help to increase the expression level of the mutant in Pichia pastoris.

Example 7: amino acid saturation mutation at position 63 of BC-PC-PLC

Mutation of the 63th asparagine of BC-PC-PLC to alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine ( F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P) ), glutamine (Q), arginine (R), threonine (T), tyrosine (V), tryptophan (W) and tyrosine (Y).

In simple terms, use the pAO-PLC vector as a template

DNA polymerase and primer pair EPPLC-1/63X-2 (X represents a single-letter abbreviation for the above 18 amino acids), and approximately 207 bp fragment was amplified by PCR. Using pAO-PLC vector as template

DNA polymerase and primer pair 63X-3/EPPLC-2 were amplified by PCR to obtain a fragment of about 576 bp. The 207 bp fragment and the approximately 576 bp fragment obtained by the previous two-step PCR were mixed as a template for the third step PCR, using the primer pair EPPLC-1/EPPLC-2 and

DNA polymerase was amplified by PCR to obtain a fragment of about 755 bp.

The 18 755 bp fragments obtained were cloned into pmAO-PLC by HindIII and EcoRI cleavage sites, respectively, to obtain 18 pmAO-PLC-N63X vectors, and 18 pmAO-PLC-N63X vectors were linearized with SalI. The 8.5 kb fragment was recovered. Competent cells of Pichia pastoris GS115 strain were prepared by LiAC method, and then 500 ng linearized 18 pmAO-PLC-N63X were transformed into GS115 competent cells by electroporation. The transformants were inoculated on MGYS plates and cultured at 30 ° C for 3 days. The monoclonal on the plate was picked, suspended in 5 μl of sterile water, and 0.5 μl was inoculated on the MM-yolk screening plate. The size of the mutant and the G15 and 1-3 white precipitation circles were compared using G15 and 1-3 as controls. The mutant positive clone PLC-N63X was used for subsequent experiments as described in Example 4.1. Among them, PLC-N63P, a mutant in which the 63th amino acid was mutated to proline, was formed without a white precipitate, indicating that the mutation inactivated the PLC.

Example 8: Shake flask fermentation enzyme activity and thermal stability of BC-PC-PLC saturated mutant strain

Take G15, 1-3, 6-6, 7-6, PLC-N63A, PLC-N63C, PLC-N63D, PLC-N63E, PLC-N63F, PLC-N63G, PLC-N63H, PLC-N63I, PLC-N63K PLC-N63L, PLC-N63M, PLC-N63P, PLC-N63Q, PLC-N63R, PLC-N63T, PLC-N63V, PLC-N63W, PLC-N63Y are first activated in liquid YPD and then inoculated in BMGY medium. Incubate overnight at 30 ° C with shaking at 220 rpm. The culture was transferred to BMMY medium, the initial OD ₆₀₀ of 6.

10 μl of the fermentation broth was added to 190 μl of PLC reaction solution (containing 0.5% soybean phospholipid, 25 mM Tris-HCl pH 7.5, 5 mM CaCl ₂ ), and incubated at 37 ° C or 60 ° C for 30 min with shaking. After the incubation, 100 μl of chloroform was added and shaken, and centrifuged at 12,000 rpm for 2 min. 80 μl of the supernatant was taken, and 20 μl of CIAP reaction solution (containing 50 mM Tris-HCl pH 9.0, 10 mM MgCl ₂ , 1 U CIAP) was added, and the mixture was incubated at 37 ° C for 1 h with shaking.

After incubation, 25 μl of the reaction was taken, and 975 μl of molybdenum blue color developing solution (containing 0.2% ascorbic acid, 0.1% ammonium molybdate) was added, and the mixture was incubated at 37 ° C for 10 min with shaking. The absorbance of the sample was measured at a wavelength of 700 nm, and the PLC activity of each fermentation broth sample was calculated.

Determination of G15, 1-3, 6-6 (PLC-N63S), 7-6, PLC-N63A, PLC-N63C, PLC-N63D, PLC-N63E, PLC-N63F, PLC-N63G, PLC-N63H with Bradford reagent , PLC-N63I, PLC-N63K, PLC-N63L, PLC-N63M, PLC-N63P, PLC-N63Q, PLC-N63R, PLC-N63T, PLC-N63V, PLC-N63W, PLC-N63Y fermentation broth protein concentration.

Thus, the specific enzyme activity of each mutant at 37 ° C and 60 ° C was obtained, as shown in FIG. 11 , it can be seen from the figure that PLC-N63P has almost no activity at 37 ° C, PLC-N63C, PLC-N63D, The specific activity of PLC-N63E and PLC-N63I was not obvious, and the specific enzyme activities of the other mutants were more than 1 times higher than that of wild type, among which PLC-N63S, PLC-N63A, PLC-N63F, PLC-N63H, PLC The specific enzyme activities of -N63K, PLC-N63R, PLC-N63T, PLC-N63W and PLC-N63Y were more than 4 times that of wild type.

When reacted at 60 °C, PLC-N63P had almost no activity. PLC-N63D and PLC-N63I had no significant effect on enzyme activity improvement. The other enzymes had more than one-fold increase in enzyme activity than wild type, among which PLC-N63S, The specific enzyme activities of PLC-N63A, PLC-N63F, PLC-N63H, PLC-N63K, PLC-N63R, PLC-N63W and PLC-N63Y were more than 6 times that of wild type.

The specific activity of the wild type at 60 ° C was 1.5 times that of the reaction at 37 ° C, while the mutants PLC-N63S, PLC-N63A, PLC-N63C, PLC-N63D, PLC-N63E, PLC-N63F, PLC- The specific enzyme activity of N63G, PLC-N63H, PLC-N63I, PLC-N63K, PLC-N63L, PLC-N63M, PLC-N63R, PLC-N63V, PLC-N63W and PLC-N63Y at 60 °C is 37 °C. 2 times or more at the time of reaction. Description Mutant PLC-N63S, PLC-N63A, PLC-N63C, PLC-N63D, PLC-N63E, PLC-N63F, PLC-N63G, PLC-N63H, PLC-N63I, PLC-N63K, PLC-N63L, PLC-N63M PLC-N63R, PLC-N63V, PLC-N63W and PLC-N63Y are more suitable for reaction at 60 °C than wild type.

After incubating all the fermentation broths at 90 ° C for 1 h, the reaction was carried out at 37 ° C and 60 ° C, respectively, to determine the specific enzyme activity after treatment at 90 ° C. As shown in Figure 12, the mutants PLC-N63F, PLC-N63W and PLC-N63Y retained 50% viability at 37 °C after treatment at 90 °C for 1 h, while the ratio of wild type to other mutants Enzyme activity is greatly reduced. As shown in Figure 13, the mutants PLC-N63F, PLC-N63W and PLC-N63Y remained viable at 30 °C after treatment at 90 °C for 1 h, while the wild type and the remaining mutants remained It is much lower than the enzyme activity. Description PLC-N63F, PLC-N63W and PLC-N63Y have good thermal stability.

As can be seen from the above examples, the 63rd amino acid of BC-PC-PLC was mutated from asparagine to alanine (A), phenylalanine (F), glycine (G), histidine (H), and Leucine (I), lysine (K), leucine (L), methionine (M), glutamine (Q), arginine (R), threonine (T), After serine (S), tyrosine (V), tryptophan (W) and tyrosine (Y), the specific enzyme activity at 37 ° C and 60 ° C was more than 2 times higher than that of the wild type. After mutation to phenylalanine (F), tryptophan (W) and tyrosine (Y) mutants showed good thermal stability after treatment at 90 ° C for 1 h and 37 ° C reaction It can retain about 50% than the enzyme activity, and the specific enzyme activity at 60 °C can retain about 30%, which is better than wild type and other mutants. In addition, the 20th arginine of BC-PC-PLC is mutated to histidine and the alanine at position 83 is mutated to aspartic acid, which can mutate the amino acid at position 63 from asparagine to serine. The amount of expression in Pichia pastoris is increased.

Sequence description

SEQ ID No: 1: wild type BC-PC-PLC DNA coding sequence;

SEQ ID No: 2: wild type BC-PC-PLC amino acid sequence;

SEQ ID No: 3: artificially synthesized a-BC-PC-PLC DNA sequence;

SEQ ID No: 4: BC-PC-PLC mutant 7-3-3 DNA coding sequence;

SEQ ID No: 5: mutant PLC-R20H DNA coding sequence;

SEQ ID No: 6: mutant PLC-R20H amino acid sequence;

SEQ ID No: 7: mutant PLC-N63S DNA coding sequence;

SEQ ID No: 8: mutant PLC-N63S amino acid sequence;

SEQ ID No: 9: mutant PLC-A83D DNA coding sequence;

SEQ ID No: 10: mutant PLC-A83D amino acid sequence;

SEQ ID No: 11: PLC-R20HN63SA83D DNA coding sequence;

SEQ ID No: 12: Amino acid sequence of PLC-R20HN63SA83D;

SEQ ID No: 13: PLC-N63A DNA coding sequence;

SEQ ID No: 14: PLC-N63A amino acid sequence;

SEQ ID No: 15: PLC-N63C DNA coding sequence;

SEQ ID No: 16: PLC-N63C amino acid sequence;

SEQ ID No: 17: PLC-N63D DNA coding sequence;

SEQ ID No: 18: PLC-N63D amino acid sequence;

SEQ ID No: 19: PLC-N63E DNA coding sequence;

SEQ ID No: 20: PLC-N63E amino acid sequence;

SEQ ID No: 21: PLC-N63F DNA coding sequence;

SEQ ID No: 22: PLC-N63F amino acid sequence;

SEQ ID No: 23: PLC-N63G DNA coding sequence;

SEQ ID No: 24: PLC-N63G amino acid sequence;

SEQ ID No: 25: PLC-N63H DNA coding sequence;

SEQ ID No: 26: PLC-N63H amino acid sequence;

SEQ ID No: 27: PLC-N63I DNA coding sequence;

SEQ ID No: 28: PLC-N63I amino acid sequence;

SEQ ID No: 29: PLC-N63K DNA coding sequence;

SEQ ID No: 30: PLC-N63K amino acid sequence;

SEQ ID No: 31: PLC-N63L DNA coding sequence;

SEQ ID No: 32: PLC-N63L amino acid sequence;

SEQ ID No: 33: PLC-N63M DNA coding sequence;

SEQ ID No: 34: PLC-N63M amino acid sequence;

SEQ ID No: 35: PLC-N63P DNA coding sequence;

SEQ ID No: 36: PLC-N63P amino acid sequence;

SEQ ID No: 37: PLC-N63Q DNA coding sequence;

SEQ ID No: 38: PLC-N63Q amino acid sequence;

SEQ ID No: 39: PLC-N63R DNA coding sequence;

SEQ ID No: 40: PLC-N63R amino acid sequence;

SEQ ID No: 41: PLC-N63T DNA coding sequence;

SEQ ID No: 42: PLC-N63T amino acid sequence;

SEQ ID No: 43: PLC-N63V DNA coding sequence;

SEQ ID No: 44: PLC-N63V amino acid sequence;

SEQ ID No: 45: PLC-N63W DNA coding sequence;

SEQ ID No: 46: PLC-N63W amino acid sequence;

SEQ ID No: 47: PLC-N63Y DNA coding sequence;

SEQ ID No: 48: PLC-N63Y amino acid sequence.

Primer sequence list

Claims

A polypeptide having phosphatidylcholine-specific phospholipase C activity, comprising the mutated amino acid sequence of SEQ ID No: 2 or an active fragment thereof, wherein the mutation comprises the amino acid represented by SEQ ID No: The asparagine at position 63 of the sequence was mutated.
The polypeptide according to claim 1, wherein the asparagine at position 63 of the amino acid sequence of SEQ ID No: 2 is mutated to serine (S), alanine (A), phenylalanine (F), group Acid (H), lysine (K), arginine (R), tryptophan (W), tyrosine (Y), cysteine (C), aspartic acid (D), Glutamate (E), glycine (G), isoleucine (I), leucine (L), methionine (M), glutamine (Q), threonine (T) or valine ( V).
The polypeptide according to claim 1 or 2, wherein the mutation further comprises the substitution of the arginine at position 20 of the amino acid sequence of SEQ ID No: 2 with histidine and the alanine at position 83 with aspartic acid. Substituting, preferably, the amino acid sequence of the polypeptide comprises the amino acid sequence shown by SEQ ID No: 12 or represented by SEQ ID No: 12.
The polypeptide according to claim 1 or 2, wherein the amino acid sequence of the polypeptide comprises a SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, The amino acid sequence of 40, 42, 44, 46 or 48 or selected from the group consisting of SEQ ID No: 8, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, The amino acid sequence composition of 44, 46 or 48.
A nucleic acid molecule encoding the polypeptide of any one of claims 1 to 4, preferably the nucleic acid molecule comprises a SEQ ID No: 7, 11, 13, 15, 17, 19, 21, 23, 25, Nucleic acid sequence of 27, 29, 31, 33, 37, 39, 41, 43, 45 or 47.
A vector comprising the nucleic acid molecule of claim 5, preferably the vector is an expression vector, more preferably, the vector is designed for expression in eukaryotic or prokaryotic cells, further preferably for bacterial cells Expression in fungal cells, yeast cells, mammalian cells, insect cells or plant cells.
A cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6, preferably a eukaryotic cell or a prokaryotic cell, more preferably a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell or a plant cell .
The phospholipase C produced by the cell of claim 7 is used.
The polypeptide according to any one of claims 1 to 4, the polypeptide encoded by the nucleic acid molecule of claim 5, or the polypeptide encoded by the vector of claim 6, or the cell of claim 7. The use of the polypeptide or the phospholipase C of claim 8 as phosphatidylcholine-specific phospholipase C is preferably used in a grease degumming process.
Use of the polypeptide of any one of claims 1 to 4, or the nucleic acid molecule of claim 5, or the vector of claim 6, or the cell of claim 7 for the preparation of a degumming enzyme.